This invention relates to a capillary liquid chromatography column comprising monodispersed particles.
The separation of molecules can be effectuated by employing liquid chromatography. A typical liquid chromatography system consists of: a column where separation of analytes is effectuated; one or more pumping units to move the solvent and sample through the column; one or more detectors to monitor the effluent exiting the column; and a data processing system used to collect and analyze data from the detector(s). A critical component of any liquid chromatography system is the column which is used to facilitate separation amongst the various analytes contained within a given sample. Liquid chromatography columns have contained within them functional chemistries which comprise the stationary phase of the column. For example, some columns have a stationary phase consisting of charged molecules, such as hydrocarbons containing an ionic moiety. In a particular instance, these ionic groups could be cations, thereby facilitating anion exchange between an anionic analyte and an anion contributed by the solvent. There are other categories of liquid chromatography columns, such as reverse phase columns. These columns typically contain a stationary phase comprising hydrocarbon functional moieties.
Analytes within a sample are introduced into a column via the solvent stream which traverses and exits the column. Based upon the chemistry of a particular analyte, and that of the stationary phase, a specific interaction between the analyte and stationary phase can occur. A critical parameter involved in this interaction is the solvent condition which provides a liquid medium carrying the sample through the column. The solvent can provide an environment which facilitates a specific interaction between an analyte and stationary phase, or it can preclude such an interaction. Those analytes that possess a relatively high affinity for the column""s stationary phase will be retained on the column, while analytes with less affinity will lightly interact with the stationary phase chemistry. Alternatively, analytes with little affinity will traverse and exit the column with minimal or no interaction with the column""s stationary phase. In a heterogenous sample, there will typically be a range of possible interactions between individual analytes and the stationary phase of a given column.
Capillary liquid chromatography is a micro-version of traditional liquid chromatography. As is true for traditional liquid chromatography, the column used in capillary liquid chromatography is of critical import. These columns typically have low solvent consumption and require low volumes of sample for analysis. These conditions translate into a higher degree of separation efficiency. Capillary liquid chromatography systems typically comprise a micro-pumping unit, a capillary column, a detector, and a data processing system.
Capillary liquid chromatography columns are typically produced using such materials as fused silica, stainless steel, or polymeric compositions. The lumen of the capillary is packed with packing material containing separation material, such as bonded silica particles. Typically, the internal diameter of the capillary column is between 50 and 500 xcexcm. Great variability exists between columns due to current manufacturing practice which significantly impacts negatively upon chromatographic reproducibility from column to column. For example, the particles used in the packing material used to pack a column are generally not monodispersed throughout the column, instead, there is typically a particle size distribution throughout the capillary column.
The present invention provides monodispersed capillary liquid chromatography columns, which increase column separation efficiency and enhance reproducibility between manufactured columns. This increase fidelity in reproducibility between capillary columns will greatly contribute to the practitioner""s ability to perform reliable and meaningful capillary chromatography.
According to the invention, the capillary column comprising monodispersed particles is formed using a suitable substrate. The capillary column is constructed of a housing having a first plate and a second plate. In the first plate a channel is formed. The dimensions (e.g. diameter) of the channel are equivalent to the dimensions (e.g. diameter) of monodispersed particle beads used to populate the channel. The channel constitutes the lumen of the capillary column. A set of indentations is positioned along the interior longitudinal axis of the capillary column""s channel at intervals corresponding to approximately half the diameter of the particle bead used to pack the column. These indentations are used in order to stabilize the positioning of the particle bead once disposed within the channel. At least one particle bead is positioned between a set of indentations. A cover plate, that is, the second plate, is positioned and bonded to the first plate in such a manner as to securely cover the lumen or channel of the first plate. In another embodiment, The first and second plate comprise an internal cavity forming a channel.
Another embodiment of the present invention includes an axial gradient capillary column. In this embodiment of the invention, particle beads of different surface chemistries are used to populate the capillary column. For example, particle beads with C18 chemistry are disposed within one or more indents located along the interior longitudinal axis of the capillary column, that is, the channel, followed by the occupancy of particle beads having C8 chemistry within one or more indents located along the same channel. Only two chemistries are used for illustrative purposes, however, more than two sets of chemistries can be employed in forming an axial gradient capillary column.
In a further embodiment of the instant invention, the diameter of the capillary column""s channel is increased to xe2x80x9cndxe2x80x9d, where xe2x80x9cnxe2x80x9d is an integer and xe2x80x9cdxe2x80x9d is the diameter of the bead. For example, if n=2, then the particle beads are placed in the channel forming a 2:1:2:1 etc. geometry. Specifically, two beads occupy a first position followed by one bead occupying a next position, etc. along the longitudinal axis of the channel of the first plate. However, as the channel increases from one dimension to two dimensions and above, with respect to bead geometry within the channel, a radial and axial gradient capillary column can be constructed. The radial gradient can be formed by applying, for example, a C8 chemistry bead next to a C18 chemistry bead perpendicular to the longitudinal axis of the channel. Such a configuration focuses the parabolic flow profile of the mobile phase to produce a flat flow profile, thereby increasing the column""s efficiency. Features of the invention include provision of a monodispersed column that is readily reproduced.